Compounds and methods for inhibiting the interaction between α-catenin and β-catenin

ABSTRACT

Modulating agents for inhibiting an interaction between α-catenin and β-catenin are provided. The modulating agents comprise one or more of: (a) a β-catenin HAV motif; (b) a peptide analogue or mimetic of a β-catenin HAV motif; or (c) an antibody or antigen-binding fragment thereof that specifically binds to a β-catenin HAV motif. Methods for using such modulating agents for inhibiting cadherin-mediated cell adhesion in a variety of contexts are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/057,363 filed Apr. 8, 1998, now U.S. Pat. No. 6,551,994, which claimsthe benefit of U.S. Provisional Application No. 60/043,361, filed onApr. 10, 1997.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to compounds and methods for usein inhibiting cadherin-mediated cell adhesion. The invention is morespecifically related to modulating agents capable of inhibiting ordisrupting interactions between α-catenin and β-catenin, and totherapeutic methods employing such agents.

2. Description of the Related Art

The ability of cells to recognize and bind to each other is afundamental property of multicellular organisms. Such recognition andbinding allows for the maintenance of tissue integrity and compartments,and prevents the inappropriate movement of cells and macromoleculesbetween tissues. Cell adhesion also contributes to the natural adhesionof synapses in the body to prevent the remodeling of synapses. Themolecules that are responsible for cellular recognition and binding arecollectively known as cell adhesion molecules (CAMs).

There are many different families of CAMs, including the immunoglobulin,integrin, selectin and cadherin superfamilies, and each cell typeexpresses a unique combination of these molecules. Cadherins are arapidly expanding family of calcium-dependent CAMs (see Munro et al.,In: Cell Adhesion and Invasion in Cancer Metastasis, P. Brodt, ed., pp.17–34, RG Landes Co. (Austin Tex., 1996)). Examples of the cadherinsuperfamily include N (neural)-cadherin, E (epithelial)-cadherin, P(placental)-cadherin, and R (retinal-cadherin). These cadherins (termedthe classical cadherins, and abbreviated CADs) are integral membraneglycoproteins that generally promote cell adhesion through homophilicinteractions (a CAD on the surface of one cell binds to an identical CADon the surface of another cell), although CADs also appear to be capableof forming heterotypic complexes with one another under certaincircumstances and with lower affinity. CADs have been shown to regulateepithelial, endothelial, neural and cancer cell adhesion, with differentCADs expressed on different cell types. CADs also regulate the formationof intercellular junctions, and consequently the establishment ofphysical and permeability barriers between tissue compartments. Ifcadherin function is abrogated, such junctions between cells do notform.

The structures of the CADs are generally similar. As illustrated in FIG.1, CADs are composed of five extracellular domains (EC1-EC5), a singlehydrophobic domain (TM) that transverses the plasma membrane (PM), andtwo cytoplasmic domains (CP1 and CP2). The first extracellular domain(EC1) contains the classical cadherin cell adhesion recognition (CAR)sequence, HAV (His-Ala-Val), along with flanking sequences on eitherside of the CAR sequence that may play a role in conferring specificity.

Inside the cell, the second cytoplasmic domain (CP2) of the classicalcadherins interacts with a cytoplasmic protein known as β-catenin (FIG.1; designated as β) (see Wheelock et al., Current Topics in Membranes43:169–185, 1996). This protein exists in a complex with anothercytoplasmic protein, known as α-catenin (FIG. 1; designated as α). Inthe absence of this β-catenin/α-catenin complex, the classical cadherinscannot promote cell adhesion. α-catenin also binds to anothercytoplasmic protein, known as α-actinin (FIG. 1; designated as ACT),which in turns interacts directly with actin-based microfilaments (FIG.1; designated as MF) of the cytoskeleton.

β-Catenin is composed of 13 domains, referred to as arm repeats (FIG. 3;see Wheelock et al., Current Topics in Membranes 43:169–185, 1996). Thearm repeat closest to the amino terminus of β-catenin (designated as thefirst arm repeat) is known to contain the α-catenin binding site. Thespecific amino acids that are directly involved in mediating theinteraction between β-catenin and α-catenin have not previously beenidentified.

Although necessary for a variety of functions in multicellularorganisms, CAM function (especially cadherin function) has beenimplicated in a range of pathological events, including the survival ofcancer cells, the migration of cancer cells (metastasis) and thevascularization of tumors (angiogenesis). In such circumstances, itwould be advantageous to modulate cadherin function. In order to developeffective therapeutic agents that modulate cadherin function, it isimportant to further understand the mechanism of cadherin-mediated celladhesion.

Accordingly, there is a need in the art for improved methods formodulating cadherin function. The present invention fulfills this needand further provides other related advantages.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for modulating cadherin-mediatedfunctions. Within certain aspects, the present invention providesmodulating agents capable of inhibiting an interaction between α-cateninand β-catenin. In one such aspect, the modulating agent comprises one ormore of: (a) the amino acid sequence KHAVV (SEQ ID NO:1); (b) a peptideanalogue or peptidomimetic of the amino acid sequence KHAVV (SEQ IDNO:1); or (c) an antibody or antigen-binding fragment thereof thatspecifically binds to a peptide comprising the amino acid sequence KHAVV(SEQ ID NO:1). Within certain embodiments, the modulating agentcomprises the sequence KHAVV (SEQ ID NO:1) within a linear peptide or acyclic peptide ring. Such modulating agents may, within certainembodiments, comprise a linear or cyclic peptide ranging from 3 to 16amino acid residues in length.

In another such aspect, a modulating agent comprises: (a) the amino acidsequence HAV; (b) a peptide analogue or peptidomimetic of the amino acidsequence HAV; or (c) an antibody or antigen-binding fragment thereofthat specifically binds to a peptide comprising the amino acid sequenceHAV; wherein the modulating agent is associated with an internalizationmoiety. In certain embodiments, the modulating agent comprises a linearor cyclic peptide sequence, which may range from 3 to 16 amino acidresidues in length. The internalization moiety may comprise, withincertain embodiments, an internalization sequence covalently linked tothe modulating agent, a liposome that encapsulates the modulating agentor an antibody or ligand that binds to a cell surface receptor.

Within further embodiments, any of the above modulating agents may belinked to a targeting agent and/or a drug.

Within other aspects, the present invention provides pharmaceuticalcompositions comprising a cell adhesion modulating agent as describedabove, in combination with a pharmaceutically acceptable carrier.

The present invention further provides, within other aspects, methodsfor disrupting an interaction between α-catenin and β-catenin in a cell,comprising contacting a cell with a cell adhesion modulating agent asdescribed above.

Within further related aspects, the present invention provides methodsfor inhibiting cellular adhesion, comprising contacting acadherin-expressing cell with a cell adhesion modulating agent asdescribed above.

In other aspects, methods are provided for treating a demyelinatingneurological disease in a mammal, comprising administering to a mammal amodulating agent as described above. The modulating agent may beadministered, within certain embodiments, by implantation with Schwanncells or by implantation with oligodendrocyte progenitor cells and/oroligodendrocytes.

The present invention further provides, within other aspects, methodsfor reducing unwanted cellular adhesion in a mammal, comprisingadministering to a mammal a modulating agent as described above.

Within further aspects, methods are provided for enhancing the deliveryof a drug through the skin of a mammal, comprising contacting epithelialcells of a mammal with a drug and a modulating agent as described above,wherein the step of contacting is performed under conditions and for atime sufficient to allow passage of the drug across the epithelialcells. In certain embodiments, the modulating agent passes into theblood stream of the mammal. The modulating agent may be linked to thedrug and/or the step of contacting may be performed via a skin patchcomprising the modulating agent and the drug.

In other aspects, methods are provided for enhancing the delivery of adrug to a tumor in a mammal, comprising administering to a mammal amodulating agent as described above. The modulating agent may beadministered to the tumor or may be administered systemically.

Within related aspects, the present invention provides methods fortreating cancer in a mammal, comprising administering to a mammal amodulating agent as described above.

The present invention further provides, within other aspects, methodsfor inhibiting angiogenesis in a mammal, comprising administering to amammal a modulating agent as described above.

Within further aspects, the present invention provides methods forenhancing drug delivery to the central nervous system of a mammal,comprising administering to a mammal a modulating agent as describedabove.

Within other aspects, the present invention provides methods forinducing apoptosis in a cadherin-expressing cell, comprising contactinga cadherin-expressing cell with a modulating agent as described above.

Within further aspects, methods are provided for modulating the immunesystem of a mammal, comprising administering to a mammal a modulatingagent as described above.

The present invention further provides, within other aspects, methodsfor preventing pregnancy in a mammal, comprising administering to amammal a modulating agent as described above.

Within other aspects, the present invention provides methods forincreasing vasopermeability in a mammal, comprising administering to amammal a modulating agent as described above.

In further aspects, methods are provided for inhibiting synapticstability in a mammal, comprising administering to a mammal a modulatingagent as described above.

Within further aspects, the present invention provides kits forenhancing transdermal drug delivery, comprising: (a) a skin patch; and(b) a modulating agent as described above. The skin patch may beimpregnated with the modulating agent and/or may further comprise adrug.

The present invention further provides polynucleotides encoding amodulating agent as described above. Such polynucleotides may beincorporated into a viral vector, such that the modulating agent isgenerated within a cell infected by the viral vector.

These and other aspects of the invention will become evident uponreference to the following detailed description and attached drawings.All references disclosed herein are hereby incorporated by reference intheir entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a schematic diagram showing the structure of a classicalcadherin and its interaction with the catenins. The five extracellulardomains are designated EC1-EC5, the hydrophobic domain that transversesthe plasma membrane (PM) is represented by TM, and the two cytoplasmicdomains are represented by CP1 and CP2. The calcium binding motifs areshown by DXNDN (SEQ ID NO:2), DXD, DVNE (SEQ ID NO: 72) and LDRE (SEQ IDNO:3). The CAR sequence, HAV, is shown within EC1. Cytoplasmic proteinsβ-catenin (β), α-catenin (α) and α-actinin (ACT), as well asmicrofilaments (MF), are also shown.

FIGS. 2A–2C illustrate the structures of representative cyclic peptidemodulating agents, comprising the representative CAR sequences CHAVC(SEQ ID NO: 34), CKHAVC (SEQ ID NO: 31), CHAVVC (SEQ ID NO: 33), CKAVVC(SEQ ID NO: 73), CHAVVNC (SEQ ID NO: 32), CKHAVVNC (SEQ ID NO: 5),CLKHAVVNC (SEQ ID NO: 23), CLKHAVVC (SEQ ID NO: 24), CLKHAVC (SEQ ID NO:30), KHAVVND (SEQ ID NO: 26), KHAVVNE (SEQ ID NO: 27), KHAVVD (SEQ IDNO: 28), KHAVVE (SEQ ID NO: 29).

FIG. 3 is a diagram showing the structure of β-catenin.

FIG. 4 illustrates the local alignment of classical cadherins withβ-catenins (SEQ ID NOs:52–71).

FIG. 5 is a Western blot of proteins immunoprecipitated from adult mousebrain extracts utilizing anti-β-catenin antibodies in the presence ofeither the representative modulating agent H-CKHAVVNC—OH (SEQ ID NO:4;Lane A) or the control peptide H-CKHGVVNC-OH (SEQ ID NO:6; Lane B) andprobed with anti-α-catenin antibodies.

FIG. 6 is a Western blot as shown in FIG. 5, re-probed withanti-β-catenin antibodies.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides methods for inhibitingcadherin-mediated cell adhesion. The present invention is based upon theidentification of an HAV motif within the first arm repeat of β-catenin(see FIG. 3) and the discovery that HAV-containing peptides are capableof disrupting interactions between α-catenin and β-catenin. Modulatingagents that comprise such peptides or antibodies directed against suchpeptides may be used to inhibit cadherin-mediated cell adhesion within avariety of contexts. For example, such agents may be used to treatdiseases or other conditions characterized by undesirable cell adhesion,to facilitate drug delivery to a specific tissue or tumor or to inhibitangiogenesis.

Modulating Agents

As noted above, the term “modulating agent,” as used herein, refers to amolecule comprising one or more of (1) a β-catenin HAV motif, (2) apeptide analogue or peptidomimetic thereof or (3) an antibody orantigen-binding fragment thereof that specifically binds to such amotif. A modulating agent is further capable of disrupting interactionsbetween α-catenin and β-catenin, as described herein.

As used herein, a “β-catenin HAV motif” comprises the tripeptidesequence HAV. Within certain preferred embodiments, the β-catenin HAVmotif further comprises at least one, and more preferably at least twoor three, amino acid residues that flank the HAV sequence in a nativeβ-catenin molecule (i.e., residues that are adjacent to the HAV sequencelocated within the first arm repeat of a native β-catenin molecule).Flanking sequences for β-catenin of a variety of organisms are shown inSEQ ID NOs:52 to 71, and FIG. 4. Flanking sequences are preferablyderived from the sequence LKHAVVNLIN (SEQ ID NO:7). Flanking residue(s)may be present on the N-terminal and/or C-terminal side of an HAV motif,preferably on both sides. Within certain preferred embodiments, amodulating agent comprises the β-catenin HAV motif KHAVV (SEQ ID NO:1).A modulating agent may consist entirely of a β-catenin HAV motif, or mayadditionally comprise further peptide and/or non-peptide regions, suchas regions that facilitate cyclization, purification or othermanipulation and/or residues having a targeting or other function.Modulating agents may further be associated (covalently ornoncovalently) with an internalization moiety, targeting agent, drug,solid support and/or detectable marker.

Modulating agents, or peptide portions thereof, may be linear or cyclicpeptides. A “linear” peptide is a peptide or salt thereof that does notcontain an intramolecular covalent bond between two non-adjacentresidues. Within preferred embodiments, linear peptide modulating agentstypically comprise from 5 to about 20 amino acid residues, preferablyfrom 5 to 16 amino acid residues and more preferably from 5 to 10 aminoacid residues. Linear peptides that may be present within a modulatingagent include, but are not limited to, KHAVVN (SEQ ID NO:8), LKHAVVN(SEQ ID NO:9), LKHAVV (SEQ ID NO:10), LKHAV (SEQ ID NO:11), KHAVV (SEQID NO:1), CKHAVVNC (SEQ ID NO:4), CLKHAVVNC (SEQ ID NO:12), CLKHAVVC(SEQ ID NO:13), CLKHAVC (SEQ ID NO:14), CKHAVVC (SEQ ID NO:15), KHAV(SEQ ID NO:16), HAVVN (SEQ ID NO:17), HAVN (SEQ ID NO:18), HAV, CKHAVC(SEQ ID NO:19), CHAVVNC (SEQ ID NO:20), CHAVVC (SEQ ID NO:21) and CHAVC(SEQ ID NO:22), as well as derivatives of the foregoing sequences havingone or more side chain modifications.

The term “cyclic peptide,” as used herein, refers to a peptide or saltthereof that comprises an intramolecular covalent bond between twonon-adjacent residues, forming a cyclic peptide ring that comprises theβ-catenin HAV motif, or analogue thereof. The intramolecular bond may bea backbone to backbone, side-chain to backbone or side-chain toside-chain bond (i.e., terminal functional groups of a linear peptideand/or side chain functional groups of a terminal or interior residuemay be linked to achieve cyclization). Preferred intramolecular bondsinclude, but are not limited to, disulfide bonds; amide bonds betweenterminal functional groups, between residue side chains or between oneterminal functional groups and one residue side chain; thioether bondsand δ₁,δ₁-ditryptophan or a derivative thereof. Preferred cyclic peptidemodulating agents generally comprise from 4 to 15 residues, morepreferably from 5 to 10 residues, within the cyclic peptide ring.Preferred cyclic peptides include CKHAVVNC (SEQ ID NO:5), CLKHAVVNC (SEQID NO:23), CLKHAVVC (SEQ ID NO:24), CKHAVVC (SEQ ID NO:25), KHAVVND (SEQID NO:26), KHAVVNE (SEQ ID NO:27), KHAVVD (SEQ ID NO:28), KHAVVE (SEQ IDNO:29), CLKHAVC (SEQ ID NO:30), CKHAVC (SEQ ID NO:31), CHAVVNC (SEQ IDNO:32), CHAVVC (SEQ ID NO:33), CHAVC (SEQ ID NO:34), KHAVD (SEQ IDNO:35) and KHAVE (SEQ ID NO:36), where the underline indicatescyclization, as well as derivatives of the foregoing sequences havingone or more side chain modifications.

As noted above, modulating agents may be polypeptides or salts thereof,containing only amino acid residues linked by peptide bonds, or mayadditionally contain non-peptide regions, such as linkers. Peptideregions of a modulating agent may comprise residues of L-amino acids,D-amino acids, or any combination thereof. Amino acids may be fromnatural or non-natural sources, provided that at least one amino groupand at least one carboxyl group are present in the molecule; α- andβ-amino acids are generally preferred. The 20 L-amino acids commonlyfound in proteins are identified herein by the conventional three-letteror one-letter abbreviations shown in Table 1.

TABLE 1 AMINO ACID ONE-LETTER AND THREE-LETTER ABBREVIATIONS A AlaAlanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C CysCysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H HisHistidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met Methionine FPhe Phenylalanine P Pro Proline S Ser Serine T Thr Threonine W TrpTryptophan Y Tyr Tyrosine V Val Valine

A modulating agent may also contain rare amino acids (such as4-hydroxyproline or hydoxylysine), organic acids or amides and/orderivatives of common amino acids, such as amino acids having theC-terminal carboxylate esterified (e.g., benzyl, methyl or ethyl ester)or amidated and/or having modifications of the N-terminal amino group(e.g., acetylation or alkoxycarbonylation), with or without any of awide variety of side-chain modifications and/or substitutions (e.g.,methylation, benzylation, t-butylation, tosylation, alkoxycarbonylation,and the like). Preferred derivatives include amino acids having aC-terminal amide group. Residues other than common amino acids that maybe present with a modulating agent include, but are not limited to,2-mercaptoaniline, 2-mercaptoproline, ornithine, diaminobutyric acid,α-aminoadipic acid, m-aminomethylbenzoic acid and α,β-diaminopropionicacid.

As noted above, a modulating agent may comprise a peptide analogue or anon-peptide peptidomimetic of a native β-catenin HAV motif, providedthat the analogue or peptidomimetic retains the ability to disrupt aninteraction between α-catenin and β-catenin. In general, a peptideanalogue of a native β-catenin sequence should retain the HAV sequence,but may contain conservative substitutions at one or more flankingresidues such that the ability to disrupt interactions between α-cateninand β-catenin is not diminished. A “conservative substitution” is one inwhich an amino acid is substituted for another amino acid that hassimilar properties, such that one skilled in the art of peptidechemistry would expect the secondary structure and hydropathic nature ofthe polypeptide to be substantially unchanged. Amino acid substitutionsmay generally be made on the basis of similarity in polarity, charge,solubility, hydrophobicity, hydrophilicity and/or the amphipathic natureof the residues. For example, negatively charged amino acids includeaspartic acid and glutamic acid; positively charged amino acids includelysine and arginine; and amino acids with uncharged polar head groupshaving similar hydrophilicity values include leucine, isoleucine andvaline; glycine and alanine; asparagine and glutamine; and serine,threonine, phenylalanine and tyrosine. Other groups of amino acids thatmay represent conservative changes include: (1) ala, pro, gly, glu, asp,gln, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile, leu, met, ala,phe; (4) lys, arg, his; and (5) phe, tyr, trp, his. The criticaldetermining feature of a peptide analogue is the ability to disrupt aninteraction between α- and β-catenin. Such an ability may be evaluatedusing the representative assays provided herein.

A peptidomimetic is a non-peptide compound that is structurally similarto a β-catenin HAV motif, such that it retains the ability to disruptinteractions between α-catenin and β-catenin, as described below.Peptidomimetics are organic compounds that mimic the three-dimensionalshape of a β-catenin HAV motif. Peptidomimetics may be designed based ontechniques that evaluate the three dimensional shape, such as nuclearmagnetic resonance (NMR) and computational techniques. NMR is widelyused for structural analysis of molecules. Cross-peak intensities innuclear Overhauser enhancement (NOE) spectra, coupling constants andchemical shifts depend on the conformation of a compound. NOE dataprovide the interproton distance between protons through space. Thisinformation may be used to facilitate calculation of the lowest energyconformation for the HAV motif. Once the lowest energy conformation isknown, the three-dimensional shape to be mimicked is known. It should beunderstood that, within embodiments described herein, an analogue orpeptidomimetic may be substituted for a native HAV motif.

Peptide modulating agents (and peptide portions of modulating agents) asdescribed herein may be synthesized by methods well known in the art,including chemical synthesis and recombinant DNA methods. For modulatingagents up to about 50 residues in length, chemical synthesis may beperformed using solution phase or solid phase peptide synthesistechniques, in which a peptide linkage occurs through the directcondensation of the α-amino group of one amino acid with the α-carboxygroup of the other amino acid with the elimination of a water molecule.Peptide bond synthesis by direct condensation, as formulated above,requires suppression of the reactive character of the amino group of thefirst and of the carboxyl group of the second amino acid. The maskingsubstituents must permit their ready removal, without inducing breakdownof the labile peptide molecule.

In solution phase synthesis, a wide variety of coupling methods andprotecting groups may be used (see Gross and Meienhofer, eds., “ThePeptides: Analysis, Synthesis, Biology,” Vol. 1–4 (Academic Press,1979); Bodansky and Bodansky, “The Practice of Peptide Synthesis,” 2ded. (Springer Verlag, 1994)). In addition, intermediate purification andlinear scale up are possible. Those of ordinary skill in the art willappreciate that solution synthesis requires consideration of main chainand side chain protecting groups and activation method. In addition,careful segment selection is necessary to minimize racemization duringsegment condensation. Solubility considerations are also a factor.

Solid phase peptide synthesis uses an insoluble polymer for supportduring organic synthesis. The polymer-supported peptide chain permitsthe use of simple washing and filtration steps instead of laboriouspurifications at intermediate steps. Solid-phase peptide synthesis maygenerally be performed according to the method of Merrifield et al., J.Am. Chem. Soc. 85:2149, 1963, which involves assembling a linear peptidechain on a resin support using protected amino acids. Solid phasepeptide synthesis typically utilizes either the Boc or Fmoc strategy.The Boc strategy uses a 1% cross-linked polystyrene resin. The standardprotecting group for α-amino functions is the tert-butyloxycarbonyl(Boc) group. This group can be removed with dilute solutions of strongacids such as 25% trifluoroacetic acid (TFA). The next Boc-amino acid istypically coupled to the amino acyl resin using dicyclohexylcarbodiimide(DCC). Following completion of the assembly, the peptide-resin istreated with anhydrous HF to cleave the benzyl ester link and liberatethe free peptide. Side-chain functional groups are usually blockedduring synthesis by benzyl-derived blocking groups, which are alsocleaved by HF. The free peptide is then extracted from the resin with asuitable solvent, purified and characterized. Newly synthesized peptidescan be purified, for example, by gel filtration, HPLC, partitionchromatography and/or ion-exchange chromatography, and may becharacterized by, for example, mass spectrometry or amino acid sequenceanalysis. In the Boc strategy, C-terminal amidated peptides can beobtained using benzhydrylamine or methylbenzhydrylamine resins, whichyield peptide amides directly upon cleavage with HF.

In the procedures discussed above, the selectivity of the side-chainblocking groups and of the peptide-resin link depends upon thedifferences in the rate of acidolytic cleavage. Orthoganol systems havebeen introduced in which the side-chain blocking groups and thepeptide-resin link are completely stable to the reagent used to removethe α-protecting group at each step of the synthesis. The most common ofthese methods involves the 9-fluorenylmethyloxycarbonyl (Fmoc) approach.Within this method, the side-chain protecting groups and thepeptide-resin link are completely stable to the secondary amines usedfor cleaving the N-α-Fmoc group. The side-chain protection and thepeptide-resin link are cleaved by mild acidolysis. The repeated contactwith base makes the Merrifield resin unsuitable for Fmoc chemistry, andp-alkoxybenzyl esters linked to the resin are generally used.Deprotection and cleavage are generally accomplished using TFA.

Those of ordinary skill in the art will recognize that, in solid phasesynthesis, deprotection and coupling reactions must go to completion andthe side-chain blocking groups must be stable throughout the entiresynthesis. In addition, solid phase synthesis is generally most suitablewhen peptides are to be made on a small scale.

Acetylation of the N-terminus can be accomplished by reacting the finalpeptide with acetic anhydride before cleavage from the resin.C-amidation may be accomplished using an appropriate resin such asmethylbenzhydrylamine resin using the Boc technology.

Following synthesis of a linear peptide, cyclization may be achieved ifdesired by any of a variety of techniques well known in the art. Withinone embodiment, a bond may be generated between reactive amino acid sidechains. For example, a disulfide bridge may be formed from a linearpeptide comprising two thiol-containing residues by oxidizing thepeptide using any of a variety of methods. Within one such method, airoxidation of thiols can generate disulfide linkages over a period ofseveral days using either basic or neutral aqueous media. The peptide isused in high dilution to minimize aggregation and intermolecular sidereactions. This method suffers from the disadvantage of being slow buthas the advantage of only producing H₂O as a side product.Alternatively, strong oxidizing agents such as I₂ and K₃Fe(CN)₆ can beused to form disulfide linkages. Those of ordinary skill in the art willrecognize that care must be taken not to oxidize the sensitive sidechains of Met, Tyr, Trp or His. Cyclic peptides produced by this methodrequire purification using standard techniques, but this oxidation isapplicable at acid pHs. Oxidizing agents also allow concurrentdeprotection/oxidation of suitable S-protected linear precursors toavoid premature, nonspecific oxidation of free cysteine.

DMSO, unlike I₂ and K₃Fe(CN)₆, is a mild oxidizing agent which does notcause oxidative side reactions of the nucleophilic amino acids mentionedabove. DMSO is miscible with H₂O at all concentrations, and oxidationscan be performed at acidic to neutral pHs with harmless byproducts.Methyltrichlorosilane-diphenylsulfoxide may alternatively be used as anoxidizing agent, for concurrent deprotection/oxidation of S-Acm, S-Tacmor S-t-Bu of cysteine without affecting other nucleophilic amino acids.There are no polymeric products resulting from intermolecular disulfidebond formation. Suitable thiol-containing residues for use in suchoxidation methods include, but are not limited to, cysteine,β,β-dimethyl cysteine (penicillamine or Pen), β,β-tetramethylenecysteine (Tmc), β,β-pentamethylene cysteine (Pmc), β-mercaptopropionicacid (Mpr), β,β-pentamethylene-β-mercaptopropionic acid (Pmp),2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline. Peptidescontaining such residues are illustrated by the following representativeformulas, in which the underlined portion is cyclized, N-acetyl groupsare indicated by N—Ac and C-terminal amide groups are represented by—NH₂:

(SEQ ID NO:37) i) H-Lys-His-Ala-Val-Val-Asn-OH (SEQ ID NO:38) ii)H-Leu-Lys-His-Ala-Val-Val-Asn-OH (SEQ ID NO:39) iii)H-His-Ala-Val-Val-Asn-OH (SEQ ID NO:40) iv) H-His-Ala-Val-Val-OH (SEQ IDNO:5) v) H-Cys-Lys-His-Ala-Val-Val-Asn-Cys-OH (SEQ ID NO:23) vi)H-Cys-Leu-Lys-His-Ala-Val-Val-Asn-Cys-OH (SEQ ID NO:32) vii)H-Cys-His-Ala-Val-Val-Asn-Cys-OH (SEQ ID NO:33) viii)H-Cys-His-Ala-Val-Val-Cys-OH (SEQ ID NO:41) ix)H-Cys-Lys-His-Ala-Val-Val-Asn-Pen-OH (SEQ ID NO:42) x)H-Tmc-Lys-His-Ala-Val-Val-Asn-Cys-OH (SEQ ID NO:43) xi)H-Pmc-Lys-His-Ala-Val-Val-Asn-Cys-OH (SEQ ID NO:44) xii)H-Mpr-Lys-His-Ala-Val-Val-Asn-Cys-OH (SEQ ID NO:45) xiii)H-Pmp-Lys-His-Ala-Val-Val-Asn-Cys-OH

It will be readily apparent to those of ordinary skill in the art that,within each of these representative formulas, any of the abovethiol-containing residues may be employed in place of one or both of thethiol-containing residues recited.

Within another embodiment, cyclization may be achieved by amide bondformation. For example, a peptide bond may be formed between terminalfunctional groups (i.e., the amino and carboxy termini of a linearpeptide prior to cyclization), with or without an N-terminal acetylgroup and/or a C-terminal amide. Within another such embodiment, thelinear peptide comprises a D-amino acid. Alternatively, cyclization maybe accomplished by linking one terminus and a residue side chain orusing two side chains, with or without an N-terminal acetyl group and/ora C-terminal amide. Residues capable of forming a lactam bond includelysine, ornithine (Orn), α-amino adipic acid, m-aminomethylbenzoic acid,α,β-diaminopropionic acid, glutamate or aspartate.

Methods for forming amide bonds are well known in the art and are basedon well established principles of chemical reactivity. Within one suchmethod, carbodiimide-mediated lactam formation can be accomplished byreaction of the carboxylic acid with DCC, DIC, EDAC or DCCl, resultingin the formation of an O-acylurea that can be reacted immediately withthe free amino group to complete the cyclization. The formation of theinactive N-acylurea, resulting from O→N migration, can be circumventedby converting the O-acylurea to an active ester by reaction with anN-hydroxy compound such as 1-hydroxybenzotriazole, 1-hydroxysuccinimide,1-hydroxynorbornene carboxamide or ethyl 2-hydroximino-2-cyanoacetate.In addition to minimizing O→N migration, these additives also serve ascatalysts during cyclization and assist in lowering racemization.Alternatively, cyclization can be performed using the azide method, inwhich a reactive azide intermediate is generated from an alkyl ester viaa hydrazide. Hydrazinolysis of the terminal ester necessitates the useof a t-butyl group for the protection of side chain carboxyl functionsin the acylating component. This limitation can be overcome by usingdiphenylphosphoryl acid (DPPA), which furnishes an azide directly uponreaction with a carboxyl group. The slow reactivity of azides and theformation of isocyanates by their disproportionation restrict theusefulness of this method. The mixed anhydride method of lactamformation is widely used because of the facile removal of reactionby-products. The anhydride is formed upon reaction of the carboxylateanion with an alkyl chloroformate or pivaloyl chloride. The attack ofthe amino component is then guided to the carbonyl carbon of theacylating component by the electron donating effect of the alkoxy groupor by the steric bulk of the pivaloyl chloride t-butyl group, whichobstructs attack on the wrong carbonyl group. Mixed anhydrides withphosphoric acid derivatives have also been successfully used.Alternatively, cyclization can be accomplished using activated esters.The presence of electron withdrawing substituents on the alkoxy carbonof esters increases their susceptibility to aminolysis. The highreactivity of esters of p-nitrophenol, N-hydroxy compounds andpolyhalogenated phenols has made these “active esters” useful in thesynthesis of amide bonds. The last few years have witnessed thedevelopment of benzotriazolyloxytris-(dimethylamino)phosphoniumhexafluorophosphonate (BOP) and its congeners as advantageous couplingreagents. Their performance is generally superior to that of the wellestablished carbodiimide amide bond formation reactions.

Within a further embodiment, a thioether linkage may be formed betweenthe side chain of a thiol-containing residue and an appropriatelyderivatized α-amino acid. By way of example, a lysine side chain can becoupled to bromoacetic acid through the carbodiimide coupling method(DCC, EDAC) and then reacted with the side chain of any of the thiolcontaining residues mentioned above to form a thioether linkage. Inorder to form dithioethers, any two thiol containing side-chains can bereacted with dibromoethane and diisopropylamine in DMF. Examples ofthiol-containing linkages are shown below:

Cyclization may also be achieved using δ₁,δ₁-Ditryptophan (i.e.,Ac-Trp-Gly-Gly-Trp-OMe) (SEQ ID NO:46), as shown below:

Representative cyclic peptide modulating agents are depicted in FIG. 2.The structures and formulas recited herein are provided solely for thepurpose of illustration, and are not intended to limit the scope of themodulating agents described herein.

For longer peptide modulating agents, recombinant methods are preferredfor synthesis. Within such methods, all or part of a modulating agentcan be synthesized in living cells, using any of a variety of expressionvectors known to those of ordinary skill in the art to be appropriatefor the particular host cell. Suitable host cells may include bacteria,yeast cells, mammalian cells, insect cells, plant cells, algae and otheranimal cells (e.g., hybridoma, CHO, myeloma). The DNA sequencesexpressed in this manner may encode portions of an endogenous β-cateninand/or other sequences. Endogenous β-catenin sequences may be preparedbased on known cDNA or genomic sequences (see Wheelock et al., CurrentTopics in Membranes 43:169–185, 1996), which may be isolated byscreening an appropriate library with probes designed based on suchknown sequences. Screens may generally be performed as described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y., 1989 (and referencescited therein). Polymerase chain reaction (PCR) may also be employed,using oligonucleotide primers in methods well known in the art, toisolate nucleic acid molecules encoding all or a portion of anendogenous β-catenin. To generate a nucleic acid molecule encoding adesired modulating agent, an endogenous β-catenin sequence may bemodified using well known techniques. For example, portions encoding oneor more HAV motifs may be joined, with or without separation byunrelated nucleic acid regions. Alternatively, portions of the desirednucleic acid sequences may be synthesized using well known techniques,and then ligated together to form a sequence encoding the modulatingagent.

As noted above, a modulating agent may comprise an antibody, orantigen-binding fragment thereof, that specifically binds to a β-cateninHAV motif. As used herein, an antibody, or antigen-binding fragmentthereof, is said to “specifically bind” to such a motif if it reacts ata detectable level (within, for example, an ELISA) with a peptidecontaining the motif, and does not react detectably with peptides thatdo not contain a β-catenin HAV motif.

Antibodies and fragments thereof may be prepared using standardtechniques. See, e.g., Harlow and Lane, Antibodies: A Laboratory Manual,Cold Spring Harbor Laboratory, 1988. In one such technique, an immunogencomprising a β-catenin HAV motif is initially injected into any of awide variety of mammals (e.g., mice, rats, rabbits, sheep or goats).Small immunogens (i.e., less than about 20 amino acids) should be joinedto a carrier protein, such as bovine serum albumin or keyhole limpethemocyanin. Following one or more injections, the animals are bledperiodically. Polyclonal antibodies specific for the β-catenin HAV motifmay then be purified from such antisera by, for example, affinitychromatography using the modulating agent or antigenic portion thereofcoupled to a suitable solid support.

Monoclonal antibodies specific for a β-catenin HAV motif may beprepared, for example, using the technique of Kohler and Milstein, Eur.J. Immunol. 6:511–519, 1976, and improvements thereto. Briefly, thesemethods involve the preparation of immortal cell lines capable ofproducing antibodies having the desired specificity from spleen cellsobtained from an animal immunized as described above. The spleen cellsare immortalized by, for example, fusion with a myeloma cell fusionpartner, preferably one that is syngeneic with the immunized animal.Single colonies are selected and their culture supernatants tested forbinding activity against the modulating agent or antigenic portionthereof. Hybridomas having high reactivity and specificity arepreferred. To evaluate the specificity of a particular antibody,conventional antigen-binding assays may be employed.

Monoclonal antibodies may be isolated from the supernatants of growinghybridoma colonies, with or without the use of various techniques knownin the art to enhance the yield. Contaminants may be removed from theantibodies by conventional techniques, such as chromatography, gelfiltration, precipitation, and extraction. Antibodies having the desiredactivity may generally be identified using immunofluorescence analysesof tissue sections, cell or other samples where the target catenin islocalized.

Within certain embodiments, the use of antigen-binding fragments ofantibodies may be preferred. Such fragments include Fab fragments, whichmay be prepared using standard techniques. Briefly, immunoglobulins maybe purified from rabbit serum by affinity chromatography on Protein Abead columns (Harlow and Lane, Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory, 1988; see especially page 309) and digested bypapain to yield Fab and Fc fragments. The Fab and Fc fragments may beseparated by affinity chromatography on protein A bead columns (Harlowand Lane, 1988, pages 628–29).

Within certain embodiments, it may be beneficial to employ modulatingagents that are associated with an internalization moiety. Aninternalization moiety is any moiety (such as a compound, liposome orparticle) that can be used to improve the ability of an agent topenetrate the lipid bilayer of the cellular plasma membrane, thusenabling the agent to readily enter the cytoplasm and disruptinteractions between cytosolic α-catenin and β-catenin. As used herein,the term “associated with” refers to covalent attachment or anon-covalent interaction mediated by, for example, ionic bonds, hydrogenbonds, van der waals forces and/or hydrophobic interactions, such thatthe internalization moiety and modulating agent remain in closeproximity under physiological conditions.

Within certain embodiments, an internalization moiety is aninternalization sequence. An internalization sequence may be anysequence (generally a peptide sequence) that is capable of facilitatingentry of the modulating agent into the cytosol of a living cell. Onesuitable internalization sequence is a 16 amino acid peptide derivedfrom the third helix of the Antennapedia protein, and having thesequence RQIKIWFQNRRMKWKK (SEQ ID NO:47; see Prochiantz, Curr. Op.Neurobiol. 6:629–34,1996) or RQIKIWPQNRRNKWKK (SEQ ID NO:48). Analoguesof this sequence (i.e., sequences having at least 25% sequence identity,such that the ability to facilitate entry into the cytosol is notdiminished) may also be employed. One such analogue is KKWKKWWKKWWKKWKK(SEQ ID NO:49). One preferred modulating agent associated with anAntennapedia internalization sequence has the sequenceKHAVVNRQIKIWFQNRRMKWKK (SEQ ID NO:50).

Alternatively, an internalization sequence may be unrelated to theAntennapedia sequence. In general, the ability of a sequence tofacilitate entry into the cytosol may be evaluated by covalently linkingsuch a sequence to a known modulating agent and evaluating the abilityof the modulating agent to disrupt interactions between α-catenin andβ-catenin, as described herein. Within such an assay, an internalizationsequence should permit a level of disruption that is statisticallygreater than that observed in the absence of internalization sequence.Preferably, an internalization sequence incorporated into a modulatingagent results in a level of disruption that is comparable to, or greaterthan, that observed for the modulating agent comprising aninternalization sequence derived from Antennapedia.

An internalization sequence may be covalently linked to a modulatingagent. Such linkage may be generated using any of a variety of meanswell known in the art, either directly or by way of a spacer. Ingeneral, spacers may be amino acid residues (e.g., amino hexanoic acid)or peptides, or may be other bi- or multi-functional compounds that canbe covalently linked to at least two peptide sequences. Covalent linkagemay be achieved via direct condensation or other well known techniques.

Other internalization moieties may also be employed. In general, anymoiety that permits a level of disruption that is statistically greaterthan that observed in its absence is considered an internalizationmoiety. Preferably, an internalization moiety results in a level ofdisruption that is comparable to, or greater than, that observed for themodulating agent associated with an internalization sequence derivedfrom Antennapedia, as described above. For example, a modulating agentmay be incorporated into a liposome (i.e., an artificial membranevesicle), using well known technology. Other internalization moietiesinclude, but are not limited to, antibodies and ligands that bind tocell surface receptors. Alternatively, a polynucleotide encoding amodulating agent may be incorporated into an appropriate viral vector,such that the modulating agent is generated within the target cell.Various particle-mediated delivery systems are also available, and theiruse is well known to those of ordinary skill in the art.

Evaluation of Modulating Agent Activity

As noted above, modulating agents are capable of disrupting aninteraction between α-catenin and β-catenin. This ability may generallybe evaluated using any suitable assay known to those of ordinary skillin the art. For example, an immunoprecipitation as described herein maybe employed. Within such an assay, disruption of the interaction ismeasured by assessing the ability of an antibody directed againstβ-catenin to immunoprecipitate α-catenin in the presence and absence ofthe modulating agent. For example, tissue such as brain may behomogenized in the presence and absence of modulating agent. The abilityof an antibody directed against β-catenin to immunoprecipitate α-cateninfrom the homogenate is then assessed by probing a Western blot ofproteins immunoprecipitated from the tissue homogenate by anti-β-cateninantibody with an anti-α-catenin antibody. The resulting signal isindicative of the level of interaction between α-catenin and β-catenin.In general, a modulating agent should inhibit such interaction by atleast 50%.

Modulating agents also inhibit cadherin-mediated cell adhesion. Thisproperty may be evaluated using any of a variety of in vitro assaysdesigned to measure the effect of the peptide on a typical cadherinresponse. The ability of an agent to modulate cell adhesion maygenerally be evaluated in vitro by assaying the effect on one or more ofthe following: (1) neurite outgrowth, (2) Schwann cell-astrocyteadhesion, (3) Schwann cell migration on astrocyte monolayers, (4)adhesion between endothelial cells, (5) adhesion between epithelialcells (e.g., normal rat kidney cells and/or human skin) and/or (6)adhesion between cancer cells. In general, a modulating agent is aninhibitor of cell adhesion if, within one or more of theserepresentative assays, contact of the test cells with the modulatingagent results in a discernible disruption of cell adhesion.

Within a representative neurite outgrowth assay, neurons may be culturedon a monolayer of cells (e.g., 3T3 fibroblasts) that express N-cadherin.Neurons grown on such cells (under suitable conditions and for asufficient period of time) extend neurites that are typically, onaverage, twice as long as neurites extended from neurons cultured on 3T3cells that do not express N-cadherin. For example, neurons may becultured on monolayers of 3T3 cells transfected with cDNA encodingN-cadherin essentially as described by Doherty and Walsh, Curr. Op.Neurobiol. 4:49–55, 1994; Williams et al., Neuron 13:583–594, 1994; Hallet al., Cell Adhesion and Commun. 3:441–450, 1996; Doherty and Walsh,Mol. Cell. Neurosci. 8:99–111, 1994; and Safell et al., Neuron18:231–242, 1997. Briefly, monolayers of control 3T3 fibroblasts and 3T3fibroblasts that express N-cadherin may be established by overnightculture of 80,000 cells in individual wells of an 8-chamber well tissueculture slide. 3000 cerebellar neurons isolated from post-natal day 3mouse brains may be cultured for 18 hours on the various monolayers incontrol media (SATO/2% FCS), or media supplemented with variousconcentrations of the modulating agent or control peptide. The culturesmay then be fixed and stained for GAP43 which specifically binds to theneurons and their neurites. The length of the longest neurite on eachGAP43 positive neuron may be measured by computer assisted morphometry.Under the conditions described above, the presence of 500 μg/mL of amodulating agent should result in a decrease in the mean neurite lengthby at least 50%, relative to the length in the absence of modulatingagent or in the presence of a negative control peptide.

The effect of a modulating agent on Schwann cell adhesion to astrocytesmay generally be evaluated using a cell adhesion assay. Briefly, Schwanncells fluorescently labeled with Di-I may be plated onto an astrocyticsurface (e.g., a glass coverslip coated with a monolayer of astrocytes)and incubated on a shaking platform (e.g., 25 rpm for 30 minutes) in thepresence and absence of modulating agent at a concentration ofapproximately 1 mg/mL. Cells may then be washed (e.g., in Hanks medium)to remove non-attached cells. The attached cells may then be fixed andcounted (e.g., using a fluorescent microscope). In general, 1 mg/mL of amodulating agent results in a decrease in cell adhesion of at least 50%.This assay evaluates the effect of a modulating agent on N-cadherinmediated cell adhesion.

Schwann cell migration may generally be evaluated using amicro-inverted-coverslip assay. In this assay, a dense Schwann cellculture is established on coverslip fragments and Schwann cell migrationaway from the fragment edge is measured. Briefly, Schwann cellsfluorescently labeled with Di-I may be plated on polylysine- andlaminin-coated fragments of a glass coverslip and allowed to bind to thesurface for 16–18 hours. Cells may then be washed (e.g., in Hanksmedium) to remove non-attached cells, and then inverted, with cellsfacing downward onto an astrocyte-coated surface. Cultures are thenincubated further for 2 days in the presence or absence of modulatingagent at a concentration of approximately 1 mg/mL and fixed. The maximummigration distance from the edge of the coverslip fragment may then bemeasured. At a level of 1 mg/mL, a modulating agent results in adecrease in the maximum migration distance of at least 50%. This assayevaluates the effect of a modulating agent on N-cadherin mediated celladhesion.

In general, the addition of a modulating agent to cells that express acadherin results in disruption of cell adhesion. A “cadherin-expressingcell,” as used herein, may be any type of cell that expresses at leastone cadherin on the cell surface at a detectable level, using standardtechniques such as immunocytochemical protocols (e.g., Blaschuk andFarookhi, Dev. Biol. 136:564–567, 1989). Cadherin-expressing cellsinclude endothelial, epithelial and/or cancer cells. For example, suchcells may be plated under standard conditions that, in the absence ofmodulating agent, permit cell adhesion. In the presence of modulatingagent (e.g., 500 μg/mL), disruption of cell adhesion may be determinedvisually within 24 hours, by observing retraction of the cells from oneanother.

For use within one such assay, bovine pulmonary artery endothelial cellsmay be harvested by sterile ablation and digestion in 0.1% collagenase(type II; Worthington Enzymes, Freehold, N.J.). Cells may be maintainedin Dulbecco's minimum essential medium supplemented with 10% fetal calfserum and 1% antibiotic-antimycotic at 37° C. in 7% CO₂ in air. Culturesmay be passaged weekly in trypsin-EDTA and seeded onto tissue cultureplastic at 20,000 cells/cm². Endothelial cultures may be used at 1 weekin culture, which is approximately 3 days after culture confluency isestablished. The cells may be seeded onto coverslips and treated (e.g.,for 30 minutes) with modulating agent or a control compound at, forexample, 500 μg/ml and then fixed with 1% paraformaldehyde. As notedabove, disruption of cell adhesion may be determined visually within 24hours, by observing retraction of the cells from one another. This assayevaluates the effect of a modulating agent on N-cadherin mediated celladhesion.

Within another such assay, the effect of a modulating agent on normalrat kidney (NRK) cells may be evaluated. According to a representativeprocedure, NRK cells (ATCC #1571-CRL) may be plated at 10–20,000 cellsper 35 mm tissue culture flasks containing DMEM with 10% FCS andsub-cultured periodically (Laird et al., J. Cell Biol. 131:1193–1203,1995). Cells may be harvested and replated in 35 mm tissue cultureflasks containing 1 mm coverslips and incubated until 50–65% confluent(24–36 hours). At this time, coverslips may be transferred to a 24-wellplate, washed once with fresh DMEM and exposed to modulating agent at aconcentration of, for example, 1 mg/mL for 24 hours. Fresh modulatingagent may then be added, and the cells left for an additional 24 hours.Cells may be fixed with 100% methanol for 10 minutes and then washedthree times with PBS. Coverslips may be blocked for 1 hour in 2% BSA/PBSand incubated for a further 1 hour in the presence of mouseanti-E-cadherin antibody (Transduction Labs, 1:250 dilution). Primaryand secondary antibodies may be diluted in 2% BSA/PBS. Followingincubation in the primary antibody, coverslips may be washed three timesfor 5 minutes each in PBS and incubated for 1 hour with donkeyanti-mouse antibody conjugated to fluorescein (diluted 1:200). Followingfurther washes in PBS (3×5 min) coverslips can be mounted and viewed byconfocal microscopy.

In the absence of modulating agent, NRK cells form characteristictightly adherent monolayers with a cobblestone morphology in which cellsdisplay a polygonal shape. NRK cells that are treated with a modulatingagent that disrupts E-cadherin mediated cell adhesion may assume anon-polygonal and elongated morphology (i.e., a fibroblast-like shape)within 48 hours of treatment with 1 mg/mL of modulating agent. Gapsappear in confluent cultures of such cells. In addition, 1 mg/mL of sucha modulating agent reproducibly induces a readily apparent reduction incell surface staining of E-cadherin, as judged by immunofluorescencemicroscopy (Laird et al., J. Cell Biol. 131:1193–1203, 1995), of atleast 75% within 48 hours.

A third cell adhesion assay involves evaluating the effect of amodulating agent on permeability of adherent epithelial and/orendothelial cell layers. For example, the effect of permeability onhuman skin may be evaluated. Such skin may be derived from a naturalsource or may be synthetic. Human abdominal skin for use in such assaysmay generally be obtained from humans at autopsy within 24 hours ofdeath. Briefly, a modulating agent (e.g., 500 μg/ml) and a test marker(e.g., the fluorescent markers Oregon Green™ and Rhodamine Green™Dextran) may be dissolved in a sterile buffer (e.g., phosphate buffer,pH 7.2), and the ability of the marker to penetrate through the skin andinto a receptor fluid (e.g., phosphate buffer) may be measured using aFranz Cell apparatus (Franz, Curr. Prob. Dermatol. 7:58–68, 1978; Franz,J. Invest. Dermatol. 64:190–195, 1975). The penetration of the markersthrough the skin may be assessed at, for example, 6, 12, 24, 36, and 48hours after the start of the experiment. In general, a modulating agentresults in a statistically significant increase in the amount of markerin the receptor compartment after 6–48 hours in the presence of 500μg/mL modulating agent. This assay evaluates the effect of a modulatingagent on E-cadherin mediated cell adhesion.

Modulating Agent Modification and Formulations

A modulating agent as described herein may, but need not, be linked toone or more additional molecules. Although modulating agents asdescribed herein may preferentially bind to specific tissues or cells,and thus may be sufficient to target a desired site in vivo, it may bebeneficial for certain applications to include an additional targetingagent. Accordingly, a targeting agent may be associated with amodulating agent to facilitate targeting to one or more specifictissues. As used herein, a “targeting agent,” may be any substance (suchas a compound or cell) that, when associated with a modulating agentenhances the transport of the modulating agent to a target tissue,thereby increasing the local concentration of the modulating agent.Targeting agents include antibodies or fragments thereof, receptors,ligands and other molecules that bind to cells of, or in the vicinityof, the target tissue. Known targeting agents include serum hormones,antibodies against cell surface antigens, lectins, adhesion molecules,tumor cell surface binding ligands, steroids, cholesterol, lymphokines,fibrinolytic enzymes and those drugs and proteins that bind to a desiredtarget site. Among the many monoclonal antibodies that may serve astargeting agents are anti-TAC, or other interleukin-2 receptorantibodies; 9.2.27 and NR-ML-05, reactive with the 250 kilodalton humanmelanoma-associated proteoglycan; and NR-LU-10, reactive with apancarcinoma glycoprotein. An antibody targeting agent may be an intact(whole) molecule, a fragment thereof, or a functional equivalentthereof. Examples of antibody fragments are F(ab′)2, -Fab′, Fab and F[v]fragments, which may be produced by conventional methods or by geneticor protein engineering. Linkage is generally covalent and may beachieved by, for example, direct condensation or other reactions, or byway of bi- or multi-functional linkers. Within other embodiments, it mayalso be possible to target a polynucleotide encoding a modulating agentto a target tissue, thereby increasing the local concentration ofmodulating agent. Such targeting may be achieved using well knowntechniques, including retroviral and adenoviral infection.

For certain embodiments, it may be beneficial to also, or alternatively,link a drug to a modulating agent. As used herein, the term “drug”refers to any bioactive agent intended for administration to a mammal toprevent or treat a disease or other undesirable condition. Drugs includehormones, growth factors, proteins, peptides and other compounds. Theuse of certain specific drugs within the context of the presentinvention is discussed below.

Within certain aspects of the present invention, one or more modulatingagents as described herein may be present within a pharmaceuticalcomposition. A pharmaceutical composition comprises one or moremodulating agents in combination with one or more pharmaceutically orphysiologically acceptable carriers, diluents or excipients. Suchcompositions may comprise buffers (e.g., neutral buffered saline orphosphate buffered saline), carbohydrates (e.g., glucose, mannose,sucrose or dextrans), mannitol, proteins, polypeptides or amino acidssuch as glycine, antioxidants, chelating agents such as EDTA orglutathione, adjuvants (e.g., aluminum hydroxide) and/or preservatives.Within yet other embodiments, compositions of the present invention maybe formulated as a lyophilizate. One or more modulating agents (alone orin combination with a targeting agent and/or drug) may, but need not, beencapsulated within liposomes using well known technology. Compositionsof the present invention may be formulated for any appropriate manner ofadministration, including for example, topical, oral, nasal,intravenous, intracranial, intraperitoneal, subcutaneous, orintramuscular administration.

A pharmaceutical composition may also, or alternatively, contain one ormore drugs, which may be linked to a modulating agent or may be freewithin the composition. Virtually any drug may be administered incombination with a modulating agent as described herein, for a varietyof purposes as described below. Examples of types of drugs that may beadministered with a modulating agent include analgesics, anesthetics,antianginals, antifungals, antibiotics, anticancer drugs (e.g., taxol ormitomycin C), antiinflammatories (e.g., ibuprofen and indomethacin),anthelmintics, antidepressants, antidotes, antiemetics, antihistamines,antihypertensives, antimalarials, antimicrotubule agents (e.g.,colchicine or vinca alkaloids), antimigraine agents, antimicrobials,antiphsychotics, antipyretics, antiseptics, anti-signaling agents (e.g.,protein kinase C inhibitors or inhibitors of intracellular calciummobilization), antiarthritics, antithrombin agents, antituberculotics,antitussives, antivirals, appetite suppressants, cardioactive drugs,chemical dependency drugs, cathartics, chemotherapeutic agents,coronary, cerebral or peripheral vasodilators, contraceptive agents,depressants, diuretics, expectorants, growth factors, hormonal agents,hypnotics, immunosuppression agents, narcotic antagonists,parasympathomimetics, sedatives, stimulants, sympathomimetics, toxins(e.g., cholera toxin), tranquilizers and urinary antiinfectives.

The compositions described herein may be administered as part of asustained release formulation (i.e., a formulation such as a capsule orsponge that effects a slow release of modulating agent followingadministration). Such formulations may generally be prepared using wellknown technology and administered by, for example, oral, rectal orsubcutaneous implantation, or by implantation at the desired targetsite. Sustained-release formulations may contain a modulating agentdispersed in a carrier matrix and/or contained within a reservoirsurrounded by a rate controlling membrane (see, e.g., European PatentApplication 710,491 A). Carriers for use within such formulations arebiocompatible, and may also be biodegradable; preferably the formulationprovides a relatively constant level of modulating agent release. Theamount of modulating agent contained within a sustained releaseformulation depends upon the site of implantation, the rate and expectedduration of release and the nature of the condition to be treated orprevented.

Pharmaceutical compositions of the present invention may be administeredin a manner appropriate to the disease to be treated (or prevented).Appropriate dosages and a suitable duration and frequency ofadministration will be determined by such factors as the condition ofthe patient, the type and severity of the patient's disease and themethod of administration. In general, an appropriate dosage andtreatment regimen provides the modulating agent(s) in an amountsufficient to provide therapeutic and/or prophylactic benefit. Withinparticularly preferred embodiments of the invention, a modulating agentor pharmaceutical composition as described herein may be administered ata dosage ranging from 0.001 to 50 mg/kg body weight, preferably from 0.1to 20 mg/kg, on a regimen of single or multiple daily doses. For topicaladministration, a cream typically comprises an amount of modulatingagent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%. Fluidcompositions typically contain about 10 ng/ml to 5 mg/ml, preferablyfrom about 10 μg to 2 mg/mL modulating agent. Appropriate dosages maygenerally be determined using experimental models and/or clinicaltrials. In general, the use of the minimum dosage that is sufficient toprovide effective therapy is preferred. Patients may generally bemonitored for therapeutic effectiveness using assays suitable for thecondition being treated or prevented, which will be familiar to those ofordinary skill in the art.

Modulating Agent Methods of Use

In general, the modulating agents and compositions described herein maybe used for modulating the adhesion of cadherin-expressing cells (i.e.,cells that express one or more of E-cadherin, N-cadherin, P-cadherin,R-cadherin and/or other cadherins, which may be known or as yetundiscovered). Such modulation may be performed in vitro and/or in vivo,preferably in a mammal such as a human.

Certain methods described herein have an advantage over prior techniquesin that they permit the passage of molecules that are large and/orcharged across barriers of cadherin-expressing cells. As described ingreater detail below, modulating agents as described herein may also beused to disrupt or enhance cell adhesion in a variety of other contexts.Within each of the methods described herein, one or more modulatingagents may generally be administered alone, or within a pharmaceuticalcomposition. In each specific method described herein, as noted above, atargeting agent may be employed to increase the local concentration ofmodulating agent at the target site.

Within one aspect, one or more modulating agents may be used for therapyof a demyelinating neurological disease in a mammal. There are a numberof demyelinating diseases, such as multiple sclerosis, characterized byoligodendrocyte death. Since Schwann cell migration on astrocytes isinhibited by N-cadherin, modulating agents that disrupt N-cadherinmediated cell adhesion as described herein, when implanted with Schwanncells into the central nervous system, may facilitate Schwann cellmigration and permit the practice of Schwann cell replacement therapy.

Multiple sclerosis patients suitable for treatment may be identified bycriteria that establish a diagnosis of clinically definite or clinicallyprobable MS (see Poser et al., Ann. Neurol. 13:227, 1983). Candidatepatients for preventive therapy may be identified by the presence ofgenetic factors, such as HLA-type DR2a and DR2b, or by the presence ofearly disease of the relapsing remitting type. Schwann cell grafts maybe implanted directly into the brain along with the modulating agent(s)using standard techniques. Suitable amounts of modulating agentgenerally range as described above, preferably from about 10 μg/mL toabout 1 mg/mL.

Alternatively, a modulating agent may be implanted with oligodendrocyteprogenitor cells (OPs) derived from donors not afflicted with thedemyelinating disease. The myelinating cell of the CNS is theoligodendrocyte. Although mature oligodendrocytes and immature cells ofthe oligodendrocyte lineage, such as the oligodendrocyte type 2astrocyte progenitor, have been used for transplantation, OPs are morewidely used. OPs are highly motile and are able to migrate fromtransplant sites to lesioned areas where they differentiate into maturemyelin-forming oligodendrocytes and contribute to repair of demyelinatedaxons (see e.g., Groves et al., Nature 362:453–55, 1993; Baron-VanEvercooren et al., Glia 16:147–64, 1996). OPs can be isolated usingroutine techniques known in the art (see e.g., Milner andFrench-Constant, Development 120:3497–3506, 1994), from many regions ofthe CNS including brain, cerebellum, spinal cord, optic nerve andolfactory bulb. Substantially greater yields of OP's are obtained fromembryonic or neonatal rather than adult tissue. OPs may be isolated fromhuman embryonic spinal cord and cultures of neurospheres established.Human fetal tissue is a potential valuable and renewable source of donorOP's for future, long range transplantation therapies of demyelinatingdiseases such as MS.

OPs can be expanded in vitro if cultured as “homotypic aggregates” or“spheres” (Avellana-Adalid et al, J. Neurosci. Res. 45:558–70,1996).Spheres (sometimes called “oligospheres” or “neurospheres”) are formedwhen OPs are grown in suspension in the presence of growth factors suchas PDGF and FGF. OPs can be harvested from spheres by mechanicaldissociation and used for subsequent transplantation or establishment ofnew spheres in culture. Alternatively, the spheres themselves may betransplanted, providing a “focal reservoir” of OPs (Avellana-Adalid etal, J. Neurosci. Res. 45:558–70, 1996).

An alternative source of OP may be spheres derived from CNS stem cells.Recently, Reynolds and Weiss, Dev. Biol. 165:1–13, 1996 have describedspheres formed from EGF-responsive cells derived from embryonicneuroepithelium, which appear to retain the pluripotentiality exhibitedby neuroepithelium in vivo. Cells dissociated from these spheres areable to differentiate into neurons, oligodendrocytes and astrocytes whenplated on adhesive substrates in the absence of EGF, suggesting thatEGF-responsive cells derived from undifferentiated embryonicneuroepithelium may represent CNS stem cells (Reynolds and Weiss, Dev.Biol. 165:1–13, 1996). Spheres derived from CNS stem cells provide analternative source of OP which may be manipulated in vitro fortransplantation in vivo. Spheres composed of CNS stem cells may furtherprovide a microenvironment conducive to increased survival, migration,and differentiation of the OPs in vivo.

The use of neurospheres for the treatment of MS may be facilitated bymodulating agents that enhance cell migration from the spheres. In theabsence of modulating agent, the cells within the spheres adhere tightlyto one another and migration out of the spheres is hindered. Modulatingagents that disrupt N-cadherin mediated cell adhesion as describedherein, when injected with neurospheres into the central nervous system,may improve cell migration and increase the efficacy of OP replacementtherapy. Neurosphere grafts may be implanted directly into the centralnervous system along with the modulating agent(s) using standardtechniques. Suitable amounts of modulating agent generally range asdescribed above, preferably from about 10 μg/mL to about 1 mg/mL.

Alternatively, a modulating agent may be administered alone or within apharmaceutical composition. The duration and frequency of administrationwill be determined by such factors as the condition of the patient, andthe type and severity of the patient's disease. Within particularlypreferred embodiments of the invention, the modulating agent orpharmaceutical composition may be administered at a dosage ranging from0.1 mg/kg to 20 mg/kg although appropriate dosages may be determined byclinical trials. Methods of administration include injection,intravenous or intrathecal (i.e., directly in cerebrospinal fluid). Amodulating agent or pharmaceutical composition may further comprise adrug (e.g., an immunomodulatory drug).

Effective treatment of multiple sclerosis may be evidenced by any of thefollowing criteria: EDSS (extended disability status scale), appearanceof exacerbations or MRI (magnetic resonance imaging). The EDSS is ameans to grade clinical impairment due to MS (Kurtzke, Neurology33:1444, 1983), and a decrease of one full step defines an effectivetreatment in the context of the present invention (Kurtzke, Ann. Neurol.36:573–79,1994). Exacerbations are defined as the appearance of a newsymptom that is attributable to MS and accompanied by an appropriate newneurologic abnormality (Sipe et al., Neurology 34:1368, 1984). Therapyis deemed to be effective if there is a statistically significantdifference in the rate or proportion of exacerbation-free patientsbetween the treated group and the placebo group or a statisticallysignificant difference in the time to first exacerbation or duration andseverity in the treated group compared to control group. MRI can be usedto measure active lesions using gadolinium-DTPA-enhanced imaging(McDonald et al. Ann. Neurol. 36:14, 1994) or the location and extent oflesions using T₂-weighted techniques. The presence, location and extentof MS lesions may be determined by radiologists using standardtechniques. Improvement due to therapy is established when there is astatistically significant improvement in an individual patient comparedto baseline or in a treated group versus a placebo group.

Efficacy of the modulating agent in the context of prevention may bejudged based on clinical measurements such as the relapse rate and EDSS.Other criteria include a change in area and volume of T2 images on MRI,and the number and volume of lesions determined by gadolinium enhancedimages.

Within other aspects, methods are provided in which cell adhesion isdiminished. In one such aspect, the present invention provides methodsfor reducing unwanted cellular adhesion by administering a modulatingagent as described herein. Unwanted cellular adhesion can occur betweentumor cells, between tumor cells and normal cells or between normalcells as a result of surgery, injury, chemotherapy, disease,inflammation or other condition jeopardizing cell viability or function.Topical administration of the modulating agent(s) is generallypreferred, but other means may also be employed. Preferably, a fluidcomposition for topical administration (comprising, for example,physiological saline) comprises an amount of modulating agent asdescribed above, and more preferably from 10 μg/mL to 1 mg/mL. Creamsmay generally be formulated as described above. Topical administrationin the surgical field may be given once at the end of surgery byirrigation of the wound or as an intermittent or continuous irrigationwith the use of surgical drains in the post-operative period or by theuse of drains specifically inserted in an area of inflammation, injuryor disease in cases where surgery does not need to be performed.Alternatively, parenteral or transcutaneous administration may be usedto achieve similar results.

Within another such aspect, methods are provided for enhancing thedelivery of a drug through the skin of a mammal. Transdermal delivery ofdrugs is a convenient and non-invasive method that can be used tomaintain relatively constant blood levels of a drug. In general, tofacilitate drug delivery via the skin, it is necessary to perturbadhesion between the epithelial cells (keratinocytes) and theendothelial cells of the microvasculature. Using currently availabletechniques, only small, uncharged molecules may be delivered across skinin vivo. The methods described herein are not subject to the same degreeof limitation. Accordingly, a wide variety of drugs may be transportedacross the epithelial and endothelial cell layers of skin, for systemicor topical administration. Such drugs may be delivered to melanomas ormay enter the blood stream of the mammal for delivery to other siteswithin the body.

To enhance the delivery of a drug through the skin, a modulating agentas described herein and a drug are contacted with the skin surface.Contact may be achieved by direct application of the modulating agent,generally within a composition formulated as a cream or gel, or usingany of a variety of skin contact devices for transdermal application(such as those described in European Patent Application No. 566,816 A;U.S. Pat. Nos. 5,613,958; 5,505,956). A skin patch provides a convenientmethod of administration (particularly for slow-release formulations).Such patches may contain a reservoir of modulating agent and drugseparated from the skin by a membrane through which the drug diffuses.Within other patch designs, the modulating agent and drug may bedissolved or suspended in a polymer or adhesive matrix that is thenplaced in direct contact with the patient's skin. The modulating agentand drug may then diffuse from the matrix into the skin. Modulatingagent(s) and drug(s) may be contained within the same composition orskin patch, or may be separately administered, although administrationat the same time and site is preferred. In general, the amount ofmodulating agent administered via the skin varies with the nature of thecondition to be treated or prevented, but may vary as described above.Such levels may be achieved by appropriate adjustments to the deviceused, or by applying a cream formulated as described above. Transfer ofthe drug across the skin and to the target tissue may be predicted basedon in vitro studies using, for example, a Franz cell apparatus, andevaluated in vivo by appropriate means that will be apparent to those ofordinary skill in the art. As an example, monitoring of the serum levelof the administered drug over time provides an easy measure of the drugtransfer across the skin.

Transdermal drug delivery as described herein is particularly useful insituations in which a constant rate of drug delivery is desired, toavoid fluctuating blood levels of a drug. For example, morphine is ananalgesic commonly used immediately following surgery. When givenintermittently in a parenteral form (intramuscular, intravenous), thepatient usually feels sleepy during the first hour, is well during thenext 2 hours and is in pain during the last hour because the blood levelgoes up quickly after the injection and goes down below the desirablelevel before the 4 hour interval prescribed for re-injection is reached.Transdermal administration as described herein permits the maintenanceof constant levels for long periods of time (e.g., days), which allowsadequate pain control and mental alertness at the same time. Insulinprovides another such example. Many diabetic patients need to maintain aconstant baseline level of insulin which is different from their needsat the time of meals. The baseline level may be maintained usingtransdermal administration of insulin, as described herein. Antibioticsmay also be administered at a constant rate, maintaining adequatebactericidal blood levels, while avoiding the high levels that are oftenresponsible for the toxicity (e.g., levels of gentamycin that are toohigh typically result in renal toxicity).

Drug delivery by the methods of the present invention also provide amore convenient method of drug administration. For example, it is oftenparticularly difficult to administer parenteral drugs to newborns andinfants because of the difficulty associated with finding veins ofacceptable caliber to catheterize. However, newborns and infants oftenhave a relatively large skin surface as compared to adults. Transdermaldrug delivery permits easier management of such patients and allowscertain types of care that can presently be given only in hospitals tobe given at home. Other patients who typically have similar difficultieswith venous catheterization are patients undergoing chemotherapy orpatients on dialysis. In addition, for patients undergoing prolongedtherapy, transdermal administration as described herein is moreconvenient than parenteral administration.

Transdermal administration as described herein also allows thegastrointestinal tract to be bypassed in situations where parenteraluses would not be practical. For example, there is a growing need formethods suitable for administration of therapeutic small peptides andproteins, which are typically digested within the gastrointestinaltract. The methods described herein permit administration of suchcompounds and allow easy administration over long periods of time.Patients who have problems with absorption through theirgastrointestinal tract because of prolonged ileus or specificgastrointestinal diseases limiting drug absorption may also benefit fromdrugs formulated for transdermal application as described herein.

Further, there are many clinical situations where it is difficult tomaintain compliance. For example, patients with mental problems (e.g.,patients with Alzheimer's disease or psychosis) are easier to manage ifa constant delivery rate of drug is provided without having to rely ontheir ability to take their medication at specific times of the day.Also patients who simply forget to take their drugs as prescribed areless likely to do so if they merely have to put on a skin patchperiodically (e.g., every 3 days). Patients with diseases that arewithout symptoms, like patients with hypertension, are especially atrisk of forgetting to take their medication as prescribed.

For patients taking multiple drugs, devices for transdermal applicationsuch as skin patches may be formulated with combinations of drugs thatare frequently used together. For example, many heart failure patientsare given digoxin in combination with furosemide. The combination ofboth drugs into a single skin patch facilitates administration, reducesthe risk of errors (taking the correct pills at the appropriate time isoften confusing to older people), reduces the psychological strain oftaking “so many pills,” reduces skipped dosage because of irregularactivities and improves compliance.

The methods described herein are particularly applicable to humans, butalso have a variety of veterinary uses, such as the administration ofgrowth factors or hormones (e.g., for fertility control) to an animal.

As noted above, a wide variety of drugs may be administered according tothe methods provided herein. Some examples of drug categories that maybe administered transdermally include anti-inflammatory drugs (e.g., inarthritis and in other condition) such as all NSAID, indomethacin,prednisone, etc.; analgesics (especially when oral absorption is notpossible, such as after surgery, and when parenteral administration isnot convenient or desirable), including morphine, codeine, Demerol,acetaminophen and combinations of these (e.g., codeine plusacetaminophen); antibiotics such as Vancomycin (which is not absorbed bythe GI tract and is frequently given intravenously) or a combination ofINH and Rifampicin (e.g., for tuberculosis); anticoagulants such asheparin (which is not well absorbed by the GI tract and is generallygiven parenterally, resulting in fluctuation in the blood levels with anincreased risk of bleeding at high levels and risks of inefficacy atlower levels) and Warfarin (which is absorbed by the GI tract but cannotbe administered immediately after abdominal surgery because of thenormal ileus following the procedure); antidepressants (e.g., insituations where compliance is an issue as in Alzheimer's disease orwhen maintaining stable blood levels results in a significant reductionof anti-cholinergic side effects and better tolerance by patients), suchas amitriptylin, imipramin, prozac, etc.; antihypertensive drugs (e.g.,to improve compliance and reduce side effects associated withfluctuating blood levels), such as diuretics and beta-blockers (whichcan be administered by the same patch; e.g., furosemide and propanolol);antipsychotics (e.g., to facilitate compliance and make it easier forcare giver and family members to make sure that the drug is received),such as haloperidol and chlorpromazine; and anxiolytics or sedatives(e.g., to avoid the reduction of alertness related to high blood levelsafter oral administration and allow a continual benefit throughout theday by maintaining therapeutic levels constant).

Numerous other drugs may be administered as described herein, includingnaturally occurring and synthetic hormones, growth factors, proteins andpeptides. For example, insulin and human growth hormone, growth factorslike erythropoietin, interleukins and inteferons may be delivered viathe skin.

Kits for administering a drug via the skin of a mammal are also providedwithin the present invention. Such kits generally comprise a device fortransdermal application (e.g., a skin patch) in combination with, orimpregnated with, one or more modulating agents. A drug may additionallybe included within such kits.

Within a related aspect, the use of modulating agents as describedherein to increase skin permeability may also facilitate sampling of theblood compartment by passive diffusion, permitting detection and/ormeasurement of the levels of specific molecules circulating in theblood. For example, application of one or more modulating agents to theskin, via a skin patch as described herein, permits the patch tofunction like a sponge to accumulate a small quantity of fluidcontaining a representative sample of the serum. The patch is thenremoved after a specified amount of time and analyzed by suitabletechniques for the compound of interest (e.g., a medication, hormone,growth factor, metabolite or marker). Alternatively, a patch may beimpregnated with reagents to permit a color change if a specificsubstance (e.g., an enzyme) is detected. Substances that can be detectedin this manner include, but are not limited to, illegal drugs such ascocaine, HIV enzymes, glucose and PSA. This technology is of particularbenefit for home testing kits.

Within a further aspect, methods are provided for enhancing delivery ofa drug to a tumor in a mammal, comprising administering a modulatingagent in combination with a drug to a tumor-bearing mammal. Preferably,the modulating agent and the drug are formulated within the samecomposition or drug delivery device prior to administration. In general,a modulating agent may enhance drug delivery to any tumor, and themethod of administration may be chosen based on the type of targettumor. For example, injection or topical administration as describedabove may be preferred for melanomas and other accessible tumors (e.g.,metastases from primary ovarian tumors may be treated by flushing theperitoneal cavity with the composition). Other tumors (e.g., bladdertumors) may be treated by injection of the modulating agent and the drug(such as mitomycin C) into the site of the tumor. In other instances,the composition may be administered systemically, and targeted to thetumor using any of a variety of specific targeting agents. Suitabledrugs may be identified by those of ordinary skill in the art based uponthe type of cancer to be treated (e.g., mitomycin C for bladder cancer).In general, the amount of modulating agent administered varies with themethod of administration and the nature of the tumor, within the typicalranges provided above, preferably ranging from about 1 μg/mL to about 2mg/mL, and more preferably from about 10 μg/mL to 1 mg/mL. Transfer ofthe drug to the target tumor may be evaluated by appropriate means thatwill be apparent to those of ordinary skill in the art. Drugs may alsobe labeled (e.g., using radionuclides) to permit direct observation oftransfer to the target tumor using standard imaging techniques.

Within a related aspect, the present invention provides methods fortreating cancer and/or inhibiting metastasis in a mammal. Cancer tumorsare solid masses of cells, growing out of control, which requirenourishment via blood vessels. The formation of new capillaries is aprerequisite for tumor growth and the emergence of metastases.Administration of modulating agents as described herein may disrupt thegrowth of such blood vessels, thereby providing effective therapy forthe cancer and/or inhibiting metastasis. Modulating agents may also beused to treat leukemias.

A modulating agent may be administered alone (e.g., via the skin) orwithin a pharmaceutical composition. For melanomas and certain otheraccessible tumors, injection or topical administration as describedabove may be preferred. For ovarian cancers, flushing the peritonealcavity with a composition comprising one or more modulating agents mayprevent metastasis of ovarian tumor cells. Other tumors (e.g., bladdertumors, bronchial tumors or tracheal tumors) may be treated by injectionof the modulating agent into the cavity. In other instances, thecomposition may be administered systemically, and targeted to the tumorusing any of a variety of specific targeting agents, as described above.In general, the amount of modulating agent administered varies dependingupon the method of administration and the nature of the cancer, but mayvary within the ranges identified above. The effectiveness of the cancertreatment or inhibition of metastasis may be evaluated using well knownclinical observations, such as the level of serum tumor markers (e.g.,CEA or PSA).

Within a further related aspect, a modulating agent may be used toinhibit angiogenesis (i.e., the growth of blood vessels frompre-existing blood vessels) in a mammal. Inhibition of angiogenesis maybe beneficial, for example, in patients afflicted with diseases such ascancer or arthritis.

The effect of a particular modulating agent on angiogenesis maygenerally be determined by evaluating the effect of the agent on bloodvessel formation. Such a determination may generally be performed, forexample, using a chick chorioallantoic membrane assay (Iruela-Arispe etal., Molecular Biology of the Cell 6:327–343, 1995). Briefly, amodulating agent may be embedded in a mesh composed of vitrogen at oneor more concentrations (e.g., ranging from about 1 to 100 μg/mesh). Themesh(es) may then be applied to chick chorioallantoic membranes. After24 hours, the effect of the modulating agent may be determined usingcomputer assisted morphometric analysis. A modulating agent shouldinhibit angiogenesis by at least 25% at a concentration of 33 μg/mesh.

The addition of a targeting agent as described above may be beneficial,particularly when the administration is systemic. Suitable modes ofadministration and dosages depend upon the condition to be prevented ortreated but, in general, administration by injection is appropriate.Dosages may vary as described above. The effectiveness of the inhibitionmay be evaluated grossly by assessing the inability of the tumors tomaintain their growth and microscopically by observing an absence ofnerves at the periphery of the tumor.

In yet another related aspect, the present invention provides methodsfor inducing apoptosis in a cadherin-expressing cell. In general,patients afflicted with cancer may benefit from such treatment.Administration may be topical, via injection or by other means, and theaddition of a targeting agent may be beneficial, particularly when theadministration is systemic. Suitable modes of administration and dosagesdepend upon the location and nature of the cells for which induction ofapoptosis is desired but, in general, dosages may vary as describedabove. A biopsy may be performed to evaluate the level of induction ofapoptosis.

The present invention also provides methods for enhancing drug deliveryto the central nervous system of a mammal. The blood/brain barrier islargely impermeable to most neuroactive agents, and delivery of drugs tothe brain of a mammal often requires invasive procedures. Using amodulating agent as described herein, however, delivery may be by, forexample, systemic administration of a modulating agent-drug-targetingagent combination, injection of a modulating agent (alone or incombination with a drug and/or targeting agent) into the carotid arteryor application of a skin patch comprising a modulating agent to the headof the patient.

In general, the amount of modulating agent administered varies with themethod of administration and the nature of the condition to be treatedor prevented, but typically varies as described above. Transfer of thedrug to the central nervous system may be evaluated by appropriate meansthat will be apparent to those of ordinary skill in the art, such asmagnetic resonance imaging (MRI) or PET scan (positron emittedtomography).

Within further aspects, modulating agents as described herein may beused for modulating the immune system of a mammal in any of severalways. Cadherins are expressed on immature B and T cells (thymocytes andbone marrow pre-B cells), as well as on specific subsets of activated Band T lymphocytes and some hematological malignancies (see Lee et al.,J. Immunol. 152:5653–5659, 1994; Munro et al., Cellular Immunol.169:309–312, 1996; Tsutsui et al., J. Biochem. 120:1034–1039, 1996;Cepek et al., Proc. Natl. Acad. Sci. USA 93:6567–6571, 1996). Modulatingagents may generally be used to modulate specific steps within cellularinteractions during an immune response or during the dissemination ofmalignant lymphocytes.

For example, a modulating agent as described herein may be used to treatdiseases associated with excessive generation of otherwise normal Tcells. Without wishing to be bound by any particular theory, it isbelieved that the interaction of cadherins on maturing T cells and Bcell subsets contributes to protection of these cells from programmedcell death. A modulating agent may decrease such interactions, leadingto the induction of programmed cell death. Accordingly, modulatingagents may be used to treat certain types of diabetes and rheumatoidarthritis, particularly in young children where the cadherin expressionon thymic pre-Tcells is greatest.

Modulating agents may also be administered to patients afflicted withcertain skin disorders (such as cutaneous lymphomas), acute B cellleukemia and excessive immune reactions involving the humoral immunesystem and generation of immunoglobulins, such as allergic responses andantibody-mediated graft rejection. In addition, patients withcirculating cadherin-positive malignant cells (e.g., during regimeswhere chemotherapy or radiation therapy is eliminating a major portionof the malignant cells in bone marrow and other lymphoid tissue) maybenefit from treatment with a modulating agent. Such treatment may alsobenefit patients undergoing transplantation with peripheral blood stemcells.

Within the above methods, the modulating agent(s) are preferablyadministered systemically (usually by injection) or topically. Amodulating agent may be linked to a targeting agent. For example,targeting to the bone marrow may be beneficial. A suitable dosage issufficient to effect a statistically significant reduction in thepopulation of B and/or T cells that express cadherin and/or animprovement in the clinical manifestation of the disease being treated.Typical dosages generally range as described above.

Within further aspects, the present invention provides methods and kitsfor preventing pregnancy in a mammal. In general, disruption ofE-cadherin function prevents the adhesion of trophoblasts and theirsubsequent fusion to form syncitiotrophoblasts. In one embodiment, oneor more modulating agents as described herein may be incorporated intoany of a variety of well known contraceptive devices, such as spongessuitable for intravaginal insertion (see, e.g., U.S. Pat. No. 5,417,224)or capsules for subdermal implantation. Other modes of administrationare possible, however, including transdermal administration, formodulating agents linked to an appropriate targeting agent.

Suitable methods for incorporation into a contraceptive device dependupon the type of device and are well known in the art. Such devicesfacilitate administration of the modulating agent(s) to the uterineregion and may provide a sustained release of the modulating agent(s).In general, modulating agent(s) may be administered via such acontraceptive device at a dosage ranging from 0.1 to 50 mg/kg, althoughappropriate dosages may be determined by monitoring hCG levels in theurine. hCG is produced by the placenta, and levels of this hormone risein the urine of pregnant women. The urine hCG levels can be assessed byradio-immunoassay using well known techniques. Kits for preventingpregnancy generally comprise a contraceptive device impregnated with oneor more modulating agents.

Alternatively, a sustained release formulation of one or more modulatingagents may be implanted, typically subdermally, in a mammal for theprevention of pregnancy. Such implantation may be performed using wellknown techniques. Preferably, the implanted formulation provides adosage as described above, although the minimum effective dosage may bedetermined by those of ordinary skill in the art using, for example, anevaluation of hCG levels in the urine of women.

The present invention also provides methods for increasingvasopermeability in a mammal by administering one or more modulatingagents or pharmaceutical compositions. Within blood vessels, endothelialcell adhesion (mediated by N-cadherin) results in decreased vascularpermeability. Accordingly, modulating agents as described herein thatdecrease N-cadherin mediated adhesion may be used to increase vascularpermeability.

Within certain embodiments, preferred modulating agents for use withinsuch methods include peptides capable of decreasing both endothelial andtumor cell adhesion. Such modulating agents may be used to facilitatethe penetration of anti-tumor therapeutic or diagnostic agents (e.g.,monoclonal antibodies) through endothelial cell permeability barriersand tumor barriers.

Treatment with a modulating agent may be appropriate, for example, priorto administration of an anti-tumor therapeutic or diagnostic agent(e.g., a monoclonal antibody or other macromolecule), an antimicrobialagent or an anti-inflammatory agent, in order to increase theconcentration of such agents in the vicinity of the target tumor,organism or inflammation without increasing the overall dose to thepatient. Modulating agents for use within such methods may be linked toa targeting agent to further increase the local concentration ofmodulating agent, although systemic administration of a vasoactive agenteven in the absence of a targeting agent increases the perfusion ofcertain tumors relative to other tissues. Suitable targeting agentsinclude antibodies and other molecules that specifically bind to tumorcells or to components of structurally abnormal blood vessels. Forexample, a targeting agent may be an antibody that binds to a fibrindegradation product or a cell enzyme such as a peroxidase that isreleased by granulocytes or other cells in necrotic or inflamed tissues.

Administration via intravenous injection or transdermal administrationis generally preferred. Effective dosages are generally sufficient toincrease localization of a subsequently administered diagnostic ortherapeutic agent to an extent that improves the clinical efficacy oftherapy of accuracy of diagnosis to a statistically significant degree.Comparison may be made between treated and untreated tumor host animalsto whom equivalent doses of the diagnostic or therapeutic agent areadministered. In general, dosages range as described above.

Within a further aspect, modulating agents as described herein may beused for controlled inhibition of synaptic stability, resulting inincreased synaptic plasticity. Within this aspect, administration of oneor more modulating agents may be advantageous for repair processeswithin the brain, as well as learning and memory, in which neuralplasticity is a key early event in the remodeling of synapses. Celladhesion molecules, particularly N-cadherin and E-cadherin, can functionto stabilize synapses, and loss of this function is thought to be theinitial step in the remodeling of the synapse that is associated withlearning and memory (Doherty et al., J. Neurobiology, 26:437–446, 1995;Martin and Kandel, Neuron, 17:567–570, 1996; Fannon and Colman, Neuron,17:423–434, 1996). Inhibition of cadherin function by administration ofone or more modulating agents may stimulate learning and memory. Forsuch aspects, administration may be via encapsulation into a deliveryvehicle such as a liposome, using standard techniques, and injectioninto, for example, the carotid artery. Alternatively, a modulating agentmay be linked to a disrupter of the blood-brain barrier. In generaldosages range as described above.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLES Example 1 Preparation of Representative Modulating Agents

This Example illustrates the solid phase synthesis of representativepeptide modulating agents.

The peptides were synthesized on a 431AApplied Biosystems peptidesynthesizer using p-Hydroxymethylphenoxymethyl polystyrene (HMP) resinand standard Fmoc chemistry. After synthesis and deprotection, thepeptides were de-salted on a Sephadex G-10 column and lyophilized. Thepeptides were analyzed for purity by analytical HPLC, and in each case asingle peak was observed. Peptides were made as stock solutions at 10 to25 mg/mL in dimethylsulfoxide (DMSO) or water and stored at −20° C.before use.

Representative peptides synthesized by this method are illustratedbelow:

Example 2 Disruption of Interactions Between α-Catenin and β-Catenin

This Example illustrates the use of representative modulating agents todisrupt interactions between α-catenin and β-catenin.

The linear peptide H-CKHAVVNC—OH (SEQ ID NO:4; Example 1, upperstructure) and the cyclic peptide H-CKHAVVNC-OH (SEQ ID NO:5; Example 1,lower structure) were synthesized using standard solid phase peptidesynthesis techniques as described above. Both of these peptides containthe amino acid sequence HAV. In addition, the cyclic peptide has adisulfide tether, Ac-Cys-S—S-Cys-NH₂.

These peptides, as well as two control peptides (H-CKHGWNC-OH, SEQ IDNO:6, and H-CKHGWNC-OH, SEQ ID NO:51) were analyzed for their ability todisrupt α-catenin/β-catenin interactions, as judged by standardimmunoprecipitation methods. FIG. 5 shows a Western blot of proteinsimmunoprecipitated from mouse brain extracts in the presence of eitherH-CKHAVVNC-OH (SEQ ID NO:4; lane A) or H-CKHGWNC-OH (SEQ ID NO:6; laneB) and probed with anti-α-catenin antibodies. Brains from adult micewere homogenized in TC buffer (10 mM Tris pH 6.8 containing 1 mM CaCl₂,500 μM phenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 μg/mlaprotinin, and 5 μg/ml pepstatin) at wet weight to volume ratio of 1:2.Five-fold concentrated IP buffer (50 mM Tris pH 7.4 containing 750 mMNaCl, 5% Triton X-100, 2.5% NP-40) was then added to the homogenate at avolume to volume ratio of 1:4. The homogenate was then incubated withcontinuous agitation for 3 hours at 4° C. in the presence of eitherH-CKHAVVNC-OH (SEQ ID NO:4) or H-CKHGVVNC-OH (SEQ ID NO:6) at aconcentration of 1 mg/ml. At the end of the incubation period, thehomogenate was centrifuged (10,000×g) for 5 minutes at 4° C. Aliquots(50 μl) of the supernatant were incubated with 1.25 μg mouse monoclonalanti-α-catenin antibody (Transduction Laboratories, Lexington, Ky.) withcontinuous agitation for 18 hours at 4° C. An aliquot (25 μl) of ProteinG Sepharose (Pharmacia Biotech, Baie d'Urfe, Quebec) suspended inone-fold concentrated IP buffer containing 500 μMphenylmethylsulfonylfluoride, 10 μg/ml leupeptin, 10 μg/ml aprotinin,and 5 μg/ml pepstatin was added to each incubation mixture and themixtures were incubated with continuous agitation for an additional 4hours at 4° C. The mixtures were then centrifuged (5,000×g) for 5minutes at 4° C. Immunoprecipitates were washed five times with one-foldconcentrated IP buffer and resuspended in solubilization buffer (62.5 mMTris pH6.8 containing 2% SDS, 10% glycerol, and 5% α-mercaptoethanol).The suspensions were heated at 100° C. for 5 minutes, and then subjectedto SDS-PAGE. Following PAGE, the proteins were electrophoreticallytransferred to nitrocellulose membrane (0.22 μm pore size; MicronSeparations Inc., Westboro, Mass.). The membrane was incubated for 1hour in TTBS buffer (25 mM Tris pH 7.6, 0.1% Tween 20 and 0.9% w/v NaCl)containing 5% w/v dry skimmed milk, and then incubated for 1 hour inTTBS containing mouse anti-α-catenin antibodies (diluted 1:500;Transduction Laboratories, Lexington, Ky.). The membrane was washed 3times with TTBS, and incubated for 45 minutes in TTBS containing goatanti-mouse IgG antibody conjugated to horseradish peroxidase (diluted1:5000; Kirkegaard and Perry Laboratories, Gaithersburg, Md.). Finally,the membrane was washed 3 times with TTBS, and the immunoreactiveproteins were detected using an enhanced chemiluminescence kit (ECL;Amersham Life Sciences Inc., Oakville, Ontario) and autoradiographicfilm (Fuji, Minami-Ashigara, Japan). Molecular mass markers (in kDa) areshown on the left-hand side of the blot. α-catenin (αCAT; molecular mass102 kDa) is indicated on the right-hand side of the blot. Mouseimmunoglobulin G was also immunoprecipitated. The mouse immunoglobulin Gheavy chain (IgG H CHAIN) is indicated on the right-hand side of theblot.

The Western blot shown in FIG. 5 was stripped and re-probed withanti-β-catenin antibodies (diluted 1:500 in TTBS; TransductionLaboratories, Lexington, Ky.). FIG. 6 shows the Western blot which wasprobed with the β-catenin antibodies. Molecular mass markers (in kDa)are shown on the left-hand side of the blot. β-catenin (βCAT; molecularmass 95 kDa) is indicated on the right-hand side of the blot. Mouseimmunoglobulin G was also immunoprecipitated. The mouse immunoglobulin Gheavy chain (IgG H CHAIN) is indicated on the right-hand side of theblot.

Only the results obtained utilizing the linear peptides H-CKHAVVNC-OH(SEQ ID NO:4) and H-CKHGVVNC-OH (SEQ ID NO:6) are shown, as similarresults were obtained using the cyclic peptides H-CKHAVVNC-OH (SEQ IDNO:5) and H-CKHGVVNH-OH (SEQ ID NO:51). In the presence of either linearor cyclic peptides containing the β-catenin HAV motif, α-catenin wasimmunoprecipitated to a lesser extent from the adult mouse brainextracts than in the presence of the control peptides. Therefore, bothlinear and cyclic peptides containing the HAV motif (amino acidsequences H-CKHAVVNC-OH (SEQ ID NO:4) and H-CKHAVVNC-OH (SEQ ID NO:5),respectively; FIG. 2) are capable of disrupting the interaction betweenα-catenin and β-catenin. None of the peptides affected the ability ofthe anti-β-catenin antibodies to immunoprecipitate β-catenin (molecularmass 95 kDa; FIG. 6).

Example 3 Disruption of Cell Adhesion

This Example illustrates the use of representative modulating agents toinhibit cadherin-mediated cell adhesion.

Three peptides (N-Ac-CKHAVVNC-NH₂, N-Ac-CKHGWNC-NH₂, and H-KHAVVN-OH;SEQ ID NOS: 5, 51 and 8 respectively) were tested for their ability todisrupt cell adhesion. Normal human breast A1N4 cells were grown ongridded glass coverslips to approximately 30% confluence. Peptides weredissolved in distilled water at a concentration of 100 μg/ml. Each ofthe three peptide solutions was mixed 1:1 with a solution containing thefluorescent marker DAPI (Molecular Probes Inc., Eugene, Oreg.) dissolvedin water at a concentration of 40 μg/ml. All of the mixtures werecentrifuged at 10,000×g for 5 minutes immediately before use in theexperiments. An aliquot (3 ml) of each peptide/DAPI mixture was taken upin an Eppendorff microinjection pipette. Microinjection was performedusing an Eppendorf microinjector and micromanipulator coupled to an IM35inverted Zeiss microscope with phase contrast and Hoffman optics. Cellsto be injected were located and their position on the grid noted. Formicroinjection, the pipette was brought close to the cell layer and themicromanipulator programmed to return to the same plane. Cells wereinjected with 0.2 pL of peptide/DAPI mixture. This corresponds toapproximately 1/100 of the cell volume and results in a finalintracellular concentration of peptide of 0.5 mg/ml. The morphology ofthe injected cells was noted at hourly intervals. After 4 hours, adifference in the morphology of the cells injected with the variouspeptides was observed. Cells injected with the peptidesN-Ac-CKHAVVNC-NH₂ (SEQ ID NO:5) and N-Ac-CKHGWNC-NH₂ (SEQ ID NO:51) wereindistinguishable from surrounding uninjected cells. In contrast, cellsinjected with the peptide H-KHAVVN-OH (SEQ ID NO:8) had become rounded,and they were detached from their neighbors. This result suggests thatthe peptide H-KHAVVN-OH (SEQ ID NO:8) is capable of disrupting theinteraction between α-catenin and β-catenin, thus causing a disruptionof cadherin-mediated cell adhesion. These results also indicate thatprotection of the C-terminus and N-terminus of a peptide renders thepeptide inactive (compare the results for N-Ac-CKHAVVNC-NH₂ (SEQ IDNO:5) in this Example with H-CKHAVVNC-OH (SEQ ID NO:5) in Example 2).

All of the above U.S. patents, U.S. patent application publications,U.S. patent applications, foreign patents, foreign patent applicationsand non-patent publications referred to in this specification and/orlisted in the Application Data Sheet, are incorporated herein byreference, in their entirety.

From the foregoing, it will be evident that although specificembodiments of the invention have been described herein for the purposeof illustrating the invention, various modifications may be made withoutdeviating from the spirit and scope of the invention.

1. A modulating agent capable of inhibiting an interaction betweenα-catenin and β-catenin, comprising one or more of: (a) the amino acidsequence KHAVV (SEQ ID NO:1); or (b) an antibody or antigen-bindingfragment thereof that specifically binds to a peptide comprising theamino acid sequence KHAVV (SEQ ID NO:1).
 2. A modulating agent accordingto claim 1, wherein the modulating agent comprises an antibody orantigen-binding fragment thereof that specifically binds to a peptidecomprising the sequence KHAVVN (SEQ ID NO:8).
 3. A modulating agentaccording to claim 1, wherein the modulating agent comprises a linear orcyclic peptide ranging from 5 to 16 amino acid residues in length.
 4. Apharmaceutical composition comprising a modulating agent comprising oneor more of: (a) the amino acid sequence KHAVV (SEQ ID NO:1); (b) apeptide analogue of the amino acid sequence KHAVV (SEQ ID NO:1) havingconservative substitutions at one or more residues flanking the aminoacid sequence HAV; or (c) an antibody or antigen-binding fragmentthereof that specifically binds to a peptide comprising the amino acidsequence KHAVV (SEQ ID NO:1); in combination with a pharmaceuticallyacceptable carrier.
 5. A method for modulating cell adhesion comprisingcontacting a cell with a modulating agent comprising one or more of: (a)the amino acid sequence KHAVV (SEQ ID NO:1); (b) a peptide analogue ofthe amino acid sequence KHAVV (SEQ ID NO:1) having conservativesubstitutions at one or more residues flanking the amino acid sequenceHAV; or (c) an antibody or antigen-binding fragment thereof thatspecifically binds to a peptide comprising the amino acid sequence KHAVV(SEQ ID NO:1).